Hepatitis E virus, known previously to cause much acute viral hepatitis in developing countries, has recently been identified as a cause of sporadic hepatitis in industrialized countries. It also has been found to cause chronic hepatitis in severely immune-compromised persons such as those receiving organ transplants. Inability to propagate the virus efficiently in cell culture or in small animal models has inhibited attempts to characterize its molecular biology or identify its pathogenic determinants. Two genotypes of the virus infect humans only, but the remaining two genotypes that infect humans are zoonotic and swine are a known reservoir. It seems certain that other animal reservoirs for human HEV must exist but they have yet to be identified and the biological factors that permit some strains to cross species boundaries are completely unknown. In FY2010 we isolated a new strain of genotype 3 HEV from the stool of a chronically infected patient and serially passaged this new virus strain in cultured human hepatoma cells and adapted it to grow more efficiently in these cells. During the adaptation process we discovered that a recombinant HEV with a 174 nucleotide insertion as well as numerous point mutations was selected. In FY2011 we constructed infectious cDNA clones of this virus from passage 1 and passage 6 and made chimeric viruses from them to identify regions important for growth in cell cultures originating from different species.We demonstrated that the 174 nucleotide insertion played a critical role in enhancing growth. We identified another insert of probable human origin when the same stool inoculum was passaged in another experiment. We demonstrated that in both cases the virus containing a foreign sequence inserted into the hypervariable region was positively selected as early as the first passage in culture. Molecular analysis of viral genomic RNA from a different chronically-infected patient confirmed the that chronic infection correlated with extensive diversification of the virus quasispecies: the hypervariable region of some virus genomes in this USA patient contained large continuous deletions and a minor portion of genomes in feces and serum had acquired a mammalian sequence which encoded 39 amino acids of S19 ribosomal protein fused to the virus nonstructural protein. Genomes with this insert were selected during virus passage in cultured cells to become the predominant species suggesting that this inserted sequence promoted virus growth. The results demonstrated that hepatitis E virus can mutate dramatically during a prolonged infection and suggest it may be important to prevent or cure chronic infections before new variants with unpredictable properties arise. In FY2011, in collaboration with XJ Meng we identified 3 amino acids that attenuated the hepatitis E swine virus for pigs and this information may prove useful for vaccine development. We also began a collaboration with Andrew Firth to determine the function of a highly conserved RNA structure in the HEV genome;introduction of synonomous mutations into this region greatly decreased infectivity of our new cDNA clone. In 2011 we developed a new ELISA assay for antibody to HEV that utilizes luciferase for readout and we identified HEV as the true cause of hepatitis in a number of US patients originally believed to have a drug-induced hepatitis. We will introduce other mutations into our cDNA clones until optimal growth is achieved and we will use these clones further to analyze the functions and interactions of the viral proteins : some of these studies will be guided by results of microarray studies recently performed on liver tissue from chimpanzees experimentally infected with HEV.